mRNA-encoded Cas13 can be used to treat dengue infections in mice.

Dengue is a major global health threat, and there are no approved antiviral agents. Prior research using Cas13 only demonstrated dengue mitigation in vitro. Here we demonstrate that systemic delivery of mRNA-encoded Cas13a and guide RNAs formulated in lipid nanoparticles can be used to treat dengue virus (DENV) 2 and 3 in mice. First, we identified guides against DENV 2 and 3 that demonstrated in vitro efficacy. Next, we confirmed that Cas13 enzymatic activity is necessary for DENV 2 or DENV 3 mitigation in vitro. Last, we show that a single dose of lipid-nanoparticle-formulated mRNA-encoded Cas13a and guide RNA, administered 1 day post-infection, promotes survival of all infected animals and serum viral titre decreases on days 2 and 3 post-infection after lethal challenge in mice. Off-target analysis in mice using RNA sequencing showed no collateral cleavage. Overall, these data demonstrate the potential of mRNA-encoded Cas13 as a pan-DENV drug.

The critical role of interleukin-6 in protection against neurotropic flavivirus infection.

West Nile virus (WNV) and Japanese encephalitis virus (JEV) are emerging mosquito-borne flaviviruses causing encephalitis globally. No specific drug or therapy exists to treat flavivirus-induced neurological diseases. The lack of specific therapeutics underscores an urgent need to determine the function of important host factors involved in flavivirus replication and disease progression. Interleukin-6 (IL-6) upregulation has been observed during viral infections in both mice and humans, implying that it may influence the disease outcome significantly. Herein, we investigated the function of IL-6 in the pathogenesis of neurotropic flavivirus infections. First, we examined the role of IL-6 in flavivirus-infected human neuroblastoma cells, SK-N-SH, and found that IL-6 neutralization increased the WNV or JEV replication and inhibited the expression of key cytokines. We further evaluated the role of IL-6 by infecting primary mouse cells derived from IL-6 knockout (IL-6-/-) mice and wild-type (WT) mice with WNV or JEV. The results exhibited increased virus yields in the cells lacking the IL-6 gene. Next, our in vivo approach revealed that IL-6-/- mice had significantly higher morbidity and mortality after subcutaneous infection with the pathogenic WNV NY99 or JEV Nakayama strain compared to WT mice. The non-pathogenic WNV Eg101 strain did not cause mortality in WT mice but resulted in 60% mortality in IL-6-/- mice, indicating that IL-6 is required for the survival of mice after the peripheral inoculation of WNV or JEV. We also observed significantly higher viremia and brain viral load in IL-6-/- mice than in WT mice. Subsequently, we explored innate immune responses in WT and IL-6-/- mice after WNV NY99 infection. Our data demonstrated that the IL-6-/- mice had reduced levels of key cytokines in the serum during early infection but elevated levels of proinflammatory cytokines in the brain later, along with suppressed anti-inflammatory cytokines. In addition, mRNA expression of IFN-α and IFN-ß was significantly lower in the infected IL-6-/- mice. In conclusion, these data suggest that the lack of IL-6 exacerbates WNV or JEV infection in vitro and in vivo by causing an increase in virus replication and dysregulating host immune response.

SARS-CoV-2 Infects Primary Neurons from Human ACE2 Expressing Mice and Upregulates Genes Involved in the Inflammatory and Necroptotic Pathways.

Transgenic mice expressing human angiotensin-converting enzyme 2 under the cytokeratin 18 promoter (K18-hACE2) have been extensively used to investigate the pathogenesis and tissue tropism of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. Neuroinvasion and the replication of SARS-CoV-2 within the central nervous system (CNS) of K18-hACE2 mice is associated with increased mortality; although, the mechanisms by which this occurs remain unclear. In this study, we generated primary neuronal cultures from K18-hACE2 mice to investigate the effects of a SARS-CoV-2 infection. We also evaluated the immunological response to SARS-CoV-2 infection in the CNS of K18-hACE2 mice and mouse neuronal cultures. Our data show that neuronal cultures obtained from K18-hACE2 mice are permissive to SARS-CoV-2 infection and support productive virus replication. Furthermore, SARS-CoV-2 infection upregulated the expression of genes involved in innate immunity and inflammation, including IFN-α, ISG-15, CXCL10, CCL2, IL-6 and TNF-α, in the neurons and mouse brains. In addition, we found that SARS-CoV-2 infection of neurons and mouse brains activates the ZBP1/pMLKL-regulated necroptosis pathway. Together, our data provide insights into the neuropathogenesis of SARS-CoV-2 infection in K18-hACE2 mice.